Poisoning with cholinesterase inhibitors remains the primary cause of generalized convulsive syndrome of toxic etiology. Substances in this group include irreversible inhibitors, primarily organophosphorus compounds (OPCs), and reversible inhibitors, such as derivatives of carbamic acid (carbamates). Carbamates and OPCs are used as components of agricultural and household chemicals (pesticides, insecticides), as well as plasticizers and polymeric materials. Carbamates are also widely applied in the pharmaceutical industry as part of medicinal products [1][2]. The number of victims of poisoning with cholinesterase inhibitors (household poisonings, agrochemical poisonings, drug overdoses, or suicides) amounts to several million per annum worldwide.

The toxic effects of cholinesterase inhibitors on the body are manifested in the development of miosis, bronchospasm, hypersecretion, vomiting, arrhythmia, and respiratory failure. These conditions result from enhanced muscarinic and nicotinic stimulation due to the inhibition of acetylcholinesterase (AChE) activity. This inhibition promotes the accumulation of acetylcholine in neuronal synapses and the development of cholinergic syndrome [3]. Additionally, butyrylcholinesterase, carboxylesterase, and some other enzymes are inhibited [1][4][5]. At the level of the central nervous system (CNS), intoxication effects include generalized convulsive seizures, which can last for more than 30 min and involve significant damage to neurons and neuroglial cells in the brain. Furthermore, cholinergic manifestations are supplemented by glutamatergic excitotoxicity, progression of neuroinflammation and neurodegeneration. Victims may experience persistent neurological disorders for an extended period of time [6].

It appears evident that a timely and effective suppression of convulsions is essential for improving the survival rates after acute poisoning with cholinesterase inhibitors, as well as for ensuring neuroprotection and prevention of CNS dysfunction in the long-term post-intoxication period. However, the existing antidote therapy is primarily aimed at reducing severity and preventing fatalities in the earliest stages of intoxication (prior to the onset of convulsions).

The effectiveness of antidote therapy decreases significantly when administered during actively developing or established convulsive syndrome. In particular, atropin [5] acts only on muscarinic, rather than on nicotinic, cholinergic receptors [6]. This compound is most effective when administered prophylactically or within the first minutes after exposure. Oximes, such as pralidoxime, etc., are recommended as prophylactic antidotes and exclusively for organophosphorus compound (OPC) poisoning [4]. Among benzodiazepines, which interact with gamma-aminobutyric acid (GABA) receptors, midazolam is preferred due to its rapid action, attributed to its high penetrative properties across the blood–brain barrier (BBB) [5]. However, their efficacy may be significantly reduced due to a substantial decline in the expression of receptor subunits and structurally associated proteins and enzymes capable of interacting with these substances within 10–20 min after exposure [6]. This greatly increases the share of refractory (antidote-resistant) convulsions.

The above-mentioned considerations underscore the necessity for developing new effective means to suppress convulsive syndrome in poisoning with cholinesterase inhibitors. One promising direction consists in the development of anticonvulsant agents based on derivatives of valproic acid. It is known that valproic acid participates in pre- and postsynaptic modulation of GABAergic signaling, affects sodium, calcium, and potassium channels, can increase extracellular levels of serotonin and dopamine in the hippocampus, modulate neurogenesis, and exert a neuroprotective effect [7]. However, the high effective dose of this anticonvulsant for generalized convulsive syndrome (over 150 mg/kg bw) limits its use as an antidote. Moreover, high doses of valproic acid are associated with a teratogenic effect [8].

With the purpose of reducing the effective dose, new pharmaceutical agents based on valproic acid are being developed. For example, valpromide (VPM), a recently developed substance, was shown to act as a prodrug (valproic acid is released during hydrolysis in the stomach) [8][9]. However, VPM, approved only in France and Italy (Depamide®), is recommended for use exclusively in bipolar disorders (not as an anticonvulsant) [9]. Valnoctamide (VCD) — an isomer of valpromide — with its own therapeutic activity (biotransformation with the release of valproic acid in the body is minimal) has been developed. Currently, VCD has successfully passed phase IIb of clinical trials and is included in the list of sedative agents as an anticonvulsant [8]. Sec-butylpropylacetamide (SPD), a recently synthesized homologue of VCD, showed high anticonvulsant activity in various experimental models in preclinical studies, including cases of benzodiazepine-resistant convulsions [8]. The efficacy of SPD is due to a significantly faster rate of penetration through the blood–brain barrier (12 times faster than valproic acid). SPD was demonstrated to preserve cognitive functions and reduce neuronal damage [10]. However, SPD and VCD are poorly soluble in water and are used in the form of an emulsion [11], which is inconvenient for use as an antidote. All the above-mentioned derivatives of valproic acid are not registered as agents for the relief of generalized convulsive syndrome.

The aim of the study is a comparative investigation of the anticonvulsant efficacy of original valproic acid aminoether substances in poisoning with a cholinesterase inhibitor — phenylcarbamate.

Original valproic acid derivatives were developed and synthesized at the Laboratory of Drug Synthesis of the Golikov Federal Research Center of Toxicology. The list and structural formulas of the tested substances are presented in Table 1.

In the experiments, outbred white male rats aged three months and weighing 200–240 g were used as the test system. The animals were obtained from the nursery of the National Research Center “Kurchatov Institute” — RAPPOLOVO Laboratory Animal Breeding Facility (Leningrad Oblast). The animals were kept under standard conditions in accordance with the rules1.

At the first stage, an assessment of acute toxicity and determination of the median lethal dose (LD50) for TVA and QVA was conducted using the express method by Prozorovsky [12]. In order to study each compound, the experimental animals were divided into four groups of two rats each; a single intraperitoneal injection of the test solutions was performed (the substances were dissolved in 0.9% sodium chloride solution). The following doses were selected for the acute toxicity study of TVA and QVA substances: QVA — 63.1, 79.4, 100.0, 126.0 mg/kg; TVA — 79.4, 100.0, 126.0, 158.0 mg/kg. Previous studies had determined the LD50 for VAA to be 170 ± 1.2 mg/kg bw [13].

At the second stage, the pharmacological activity of the substances was studied using a model of convulsions induced by a reversible cholinesterase inhibitor — substituted 2[(dimethylamino)methyl] aryldimethylcarbamate hydrochloride (hereinafter referred to as phenylcarbamate) [14]. Phenylcarbamate was administered as a single intraperitoneal injection at a dose of 1 mg/kg bw [15]. The following experimental groups were formed: a group with isolated administration of phenylcarbamate “P” as the convulsive agent (n = 8), and three groups with administration of “P” followed by administration of the test corrective agents: P+VAA (n = 16), P+TVA (n = 16), P+QVA (n = 16). The anticonvulsant efficacy of the VAA, QVA, and TVA substances was assessed at two doses — 21.5 mg/kg bw and 43.0 mg/kg bw (eight animals per dose for each test substance).

The selection of effective doses of the test substances and the administration regimen was based on the dosage and usage protocols of sodium valproate (Convulex®) adopted in clinical practice for humans. The average dose of 7 mg/kg bw was used as the basis for calculating the effective dose. Interspecies dose conversion from human to rat was performed using the standard recommendations of Mironov2. The test dose values were calculated using the following coefficients: the therapeutic dose for rats was 7.0 × 39 (coefficient for a human weighing 70 kg) / 6.5 (coefficient for a rat weighing 200 g) ≈ 43.0 mg/kg bw.

The test substances were dissolved in a 0.9% sodium chloride solution and administered intraperitoneally, taking interspecies dose conversion into account. The volume of the intraperitoneally administered solution was 0.1 mL/100 g. Administration was performed within the first minutes after the onset of seizures at levels 3–4 on the Racine scale [16], which were induced by the administration of the cholinesterase inhibitor.

The following efficacy indicators were taken into account: latent period, severity and duration of convulsive syndrome, and mortality. Observation and recording of lethal outcomes were conducted over a 24-h period. Mortality was assessed based on the proportion of deceased rats relative to their total number in the study group after the administration of the convulsive agent during the 24-h observation period. The severity of convulsive syndrome in the experiment was assessed using the Racine scale. Seizures of level 4 and above, equivalent to generalized clonic-tonic seizures in humans, were classified as severe. The duration of convulsive syndrome was measured in minutes.

To assess the significance of differences in the frequency of rats exhibiting severe seizures, Fisher’s exact test was used. A comparative assessment of the convulsive syndrome indicators in male rats poisoned with phenylcarbamate was performed using the Kruskal–Wallis test. To identify differences between individual groups, as well as between the studied substances and the “P” group, Dunn’s multiple comparison test (post-hoc analysis) was employed. Statistical processing of the research results was carried out using the Statistica 13.0 software package (Statsoft, USA).

In the course of work to determine the quantitative characteristics of acute toxicity upon intraperitoneal administration of QVA, the following distribution of rat mortality in each dose subgroup was established: 0, 0, 2, 2 individuals, which allowed for the determination of the LD50 for QVA at a level of 89.8 ± 7.1 mg/kg; upon intraperitoneal administration of TVA — 0, 0, 2, 2 individuals, which corresponded to an LD50 of 113.1 ± 8.9 mg/kg. Based on the obtained LD50 values of the original valproic acid aminoethers, the compounds can be classified as class 3 moderately toxic substances.

The experimental model of seizures induced by a reversible acetylcholinesterase inhibitor (phenylcarbamate) [12][13] following the administration of VAA, QVA, and TVA at two doses (21.5 mg/kg bw and 43.0 mg/kg bw) revealed no statistically significant difference between the share of deceased rats in the group of animals receiving the convulsive agent, regardless of the dose and the recorded time interval. During the experiment, the death of one animal in the “P” group at the 30-min observation mark, the death of one rat following the administration of TVA at a dose of 21.5 mg/kg bw at the 10-min observation mark, and the death of three rats in the QVA group at a dose of 43.0 mg/kg bw at the 20-min observation mark were recorded.

After the administration of phenylcarbamate, the number of rats with severe convulsions in the “P” group began to decrease from the 30-min observation point by 14%; by 70 min, convulsions were no longer recorded in the animals.

The administration of the original aminoethers reduced the number of animals with convulsion severity of level 4 and above (Racine scale), which corresponds to a generalized convulsive syndrome in humans (Table 2).

Following the administration of the test substances at a dose of 21.5 mg/kg bw, a statistically significant reduction in the proportion of rats with severe convulsions was observed during the 10–40 min period of observation (Table 2).

In the P+QVA group, animals with severe convulsions were completely absent starting from the 10-min observation mark, whereas in the other two groups, the anticonvulsant effect occurred later. Specifically, in the P+TVA group, no severe convulsions were observed at 10 min; however, cases of clonic-tonic convulsions were recorded over the next 20 min, which ceased by 40 min. In the P+VAA group, a reduction in convulsion severity occurred by the 20-min observation mark.

No statistically significant intergroup differences were identified when comparing animals that received different substances at the same time interval.

When the substances were administered at a dose of 43.0 mg/kg bw, the anticonvulsant efficacy in the P+VAA and P+TVA groups generally coincided with that established at the lower dose. However, in the P+QVA group, 10 min after substance administration, the number of animals with severe convulsions did not decrease compared to the “P” group. At the same time, by 10 min, in the P+VAA and P+TVA groups, the number of animals with convulsive syndrome was statistically significantly lower than that in the group with isolated administration of phenylcarbamate.

When assessing the total duration of convulsions using statistical processing of the results with the Kruskal–Wallis test, the presence of statistically significant differences between all studied groups upon administration of the substances at both doses was revealed (Table 3).

When constructing a quadratic matrix of post-hoc comparisons (p-values) for the duration of convulsions following intraperitoneal administration of the test substances and in the phenylcarbamate group using Dunn’s post-hoc test, no statistically significant intergroup differences were identified. However, when comparing the duration of convulsions in the P+QVA group (dose 21.5 mg/kg bw) with the values in the “P” group, a statistically significant reduction in the median (Me) duration of convulsions by 77% was detected (p = 0.001). A lower median duration of convulsions by 57% was also recorded when comparing the indicator in the P+QVA and P+VAA groups at the dose level of 21.5 mg/kg bw (p = 0.041).

When the substances were administered at a dose of 43.0 mg/kg bw, differences in the duration of convulsions were established, comparable to the results obtained when studying the effects of lower doses. Specifically, significant differences were found when comparing the P+QVA group with the phenylcarbamate group (p = 0.001) and in the intergroup comparison between P+QVA and P+VAA (p = 0.039).

Time-weighted sums of seizure intensity scores (area under the curve “seizure score–time,” AUC) were calculated. Deceased individuals were excluded from the calculations. It was demonstrated (Table 4) that there are statistically significant differences between the AUC indicators upon administration of the substances at doses of 21.5 mg/kg bw (p = 0.001) and 43.0 mg/kg bw (p = 0.001).

Upon administration of the substances at a dose of 21.5 mg/kg bw, the time-weighted sums of seizure intensity scores in the P+VAA group were found to be statistically significantly lower by 77% than the corresponding indicator in the phenylcarbamate group (p = 0.041), and in the P+QVA group — by 93% (p = 0.001) based on the median, respectively. In the P+TVA group, the AUC level was lower by 84%, although not reaching statistical significance compared to the “P” group. A statistically significant decrease in the AUC indicator was also recorded in the P+QVA group compared to the P+TVA group, with a difference of 78% (p = 0.026).

When the substances were administered at a dose of 43.0 mg/kg bw, a significant reduction in the median AUC indicator was noted: by 59% (p = 0.015) in the P+VAA group and by 79% in the P+TVA group (p = 0.001) compared to the animals that received only phenylcarbamate.

Figures 1 and 2 show the dynamics of the median scores for the severity of convulsive syndrome (from 0 to 6 points on the Racine scale) after administration of the test substances over a 100-min observation period. Administration of the QVA substance at a dose of 21.5 mg/kg bw demonstrated the most favorable time course for the severity of convulsive syndrome, corresponding to the greater anticonvulsant efficacy of this compound. Conversely, when a dose of 43.0 mg/kg bw was administered, more preferable changes were observed in animals that received VAA and TVA.

Our results indicate that the administration of original valproic acid aminoethers in the setting of acute intoxication with a cholinesterase inhibitor contributed to a pronounced reduction in the duration of convulsions within the first 10–20 min after the onset of exposure. The highest efficacy was demonstrated by QVA at a dose of 21.5 mg/kg bw. This dose led to cessation of convulsions in 100% of animals after 10 min.

The time-weighted sum of seizure intensity scores (AUC) in the groups receiving the original valproic acid aminoethers at both doses was significantly lower compared to the group of animals without therapy (only phenylcarbamate administration), indicating sufficient efficacy of the test substances in reducing the severity of not only intense but also other types of convulsions. Overall, the lowest median AUC value (corresponding to the highest efficacy) was established for QVA (valproic acid quinuclidinol aminoether) when administered at a dose of 21.5 mg/kg bw. The VAA and TVA samples at the studied doses were comparable to each other in terms of efficacy.

Although no statistically significant differences in mortality were detected between the studied groups across all time intervals, the identified death cases of three animals at the 20-min mark of the experiment following the use of QVA at a high dose (43.0 mg/kg bw) require further investigation. It is evident that these cases could have been associated with either the toxicity of QVA or the individual reaction of specific animals to phenylcarbamate exposure, independent of QVA administration. Furthermore, the pronounced efficacy of QVA at a dose of 21.5 mg/kg bw suggests that a further dose reduction without efficacy loss is possible and that the 43 mg/kg bw dose may be excessive. It has been previously established that the anticonvulsant efficacy of valpromide in experiments on mice is 3–5 times higher than that of valproic acid [9]; it is possible that in the case of QVA, the optimal dose will also be significantly lower and not associated with any manifestations oftoxicity.

Previous animal models demonstrated that, in comparision with atropine — a standard antidote for intoxication with cholinesterase inhibitors [17], VAA is more effective in reducing the duration of convulsive syndrome. In this light, the developed original valproic acid derivatives should be considered promising for further study and the development of agents to terminate the convulsive syndrome induced by cholinesterase inhibitors, including irreversible ones.

1 SP 2.2.1.3218-14 dated September 28, 2014 “Sanitary and Epidemiological Requirements for the Design, Equipment, and Maintenance of Experimental-Biological Clinics (Vivaria)”. 2 Mironov AN. Guidelines for conducting preclinical drug trials. Part 1. Moscow: Grif&K; 2012.